Diagnostic dry reagent analytical devices are common products used in clinical settings for urinalysis and blood testing, particularly glucose monitoring. Results are obtained instrumentally or visually as thresholds and quantitative outputs. Dry reagent analytical devices typically involve absorbent pads containing dispersed reagent systems which react with analytes (components to be detected) in fluid test samples applied to the device to provide a detectable response. These reagents contain indicator dyes, metals, enzymes, polymers, antibodies and various other chemicals dried onto carriers. Carriers often used are papers, membranes or polymers with various sample uptake and transporting properties.
Some reagent strips use only one reagent area to contain all chemicals needed to generate color response to the analyte. In some cases, up to five competing and timed chemical reactions are occurring within one reagent layer. Hemastix® reagent strips (Bayer), a method for detecting blood in urine, is an example of multiple chemical reactions occurring in a single reagent. The analyte detecting reaction is based on the peroxidase-like activity of hemoglobin that catalyzes the oxidation of a indicator, 3,3′,5,5′-tetramethyl-benzidine, by diisopropylbenzene dihydroperoxide. In the same pad, a second reaction occurs to remove ascorbic acid interference, based on the catalytic activity of a ferric-HETDA complex that catalyzes the oxidation of ascorbic acid by diisopropylbenzene dihydroperoxide.
Typical chemical reactions occurring in dry reagent strips can be grouped as dye binding, enzymatic, immunological, and REDOX catalysis. Dye binding to analytes such as albumin leads to color changes at micromolar levels. Indicator dyes can be covalently bound to the analyte (diazonium compounds binding bilirubin) or tightly associated to the analyte (sodium sensing indicators). Enzymatic reactions can be used for the detection of enzymes at micromolar levels through reactions with color forming substrates. Enzymatic reactions can also be used for the detection of molecules, such as glucose, through reactions with enzymes to yield colored end products. Particle labeled antibodies are the primary reagents that provide for the detectable reaction of immunologic strips based on chromatography. REDOX catalysis involves the use of metal chelates to oxidize or reduce indicators in the presence of specific analytes such as hemoglobin and can detect molecules down to the nanomolar level. Certain of these devices involve an enzymatic reaction with the analyte in the presence of a peroxidase and a hydroperoxide to cause a detectable color change in a redox dye and are normally based on the use of filter paper as the absorbent pad.
Dry reagent device designs often include multiple reagent layers to measure one analyte. This change allowed chemical reagent systems to be placed into distinct reagent layers and provided for reaction separation steps such as chromatography and filtration. Immuno-chromatography strips are constructed with chemical reactions occurring a distinct layers of reagents. The CLINITEST® hCG strip test (Bayer) for human chorionic gonadotropin is an example of a dry reagent strip test with four reagent layers. The first layer at the tip of the strip is for sample application and overlaps the next reagent layer, providing for transfer of the patient sample (urine) to the first reagent area. The treated sample then migrates across a third layer, where reactants are immobilized for color development. This migration is driven by a fourth pad that takes up the excess specimen. The chromatography reaction takes place in the third layer, called the test or capture zone, typically a nitrocellulose membrane. In the first and second layers, an analyte specific antibody reacts with the analyte in the specimen and is chromatographically transferred to the nitrocellulose membrane. The antibody is bound to colored latex particles as a label. If the sample contains the analyte, it reacts with the labeled antibody. In the capture zone, a second antibody is immobilized in a band and captures particles when analyte is present. A colored test line is formed. A second band of reagent is also immobilized in the capture zone to allow a control line to react with particles, forming color. Color at the control line is always formed when the test system is working properly, even in the absence of hCG in the patient sample.
Whole blood glucose strips often use multiple reagents to trap intact red blood cells that interfere with the color generation layer. One example is GLUCOMETER Encore® (Bayer), which uses a trapping layer placed directly over the color-generating layer. The color is read from the bottom of the strip through a transparent window. Other designs allow the sample to migrate to a color-generating layer aside from the trapping layer and color is read from the top of the strip. Whole blood test strips often use plastic cassettes to hold the reaction layers in place. Multiple layers of reagent have also been applied to film slides such as the reagent system used with the Ektachem analyzer (Vitros) developed by Eastman Kodak Company (1980). Slides were able to use multiple separating, spreading and color forming layers to enhance colors.
These dry reagent devices are inexpensive and convenient to use but suffer from certain limitations. For example, immunoassays require separation of ingredients to operate, which is often achieved by protein binding. Migration of reagents and analytes often presents problems, leading to inaccurate results. The connections between layers are critical to obtaining accurate results and often fluid transfer between these layers is difficult to control. In the dry reagent format, such as that described by Greenquist in U.S. Pat. No. 4,806,311, an analyte is bound to a labeled reagent and then passed to a detection zone where the amount of the analyte is measured by the amount of labeled reagent. Unreacted labeled reagent is immobilized by immobilized analyte in the reagent zone. Any labeled reagent-analyte which passes into the detection zone is prevented from back migration by being immobilized in the detection zone.
The assembly and fabrication of multilayered devices has not been completely successful. In EP 0226 465 A2 and U.S. Pat. No. 3,992,158 for example, films have been used to separate layers of reagents. However, these devices require tight control of the pore size and shape and of the thickness of the films. One consequence of such designs is that the reagents cannot be on filter paper, since such papers do not have the well defined pore structures of films or the uniform surfaces needed for uniform thickness. But, filter paper is desirable in multilayered devices since they are well suited for use with many reagents due to their inert nature and high water absorbtivity. Thus, filter paper has been used, along with a nylon mesh covering. Such devices rely on surface contact between the reagent layers and this causes reagents to mix on the surface into one layer. The present invention avoids this result and keeps the reagents in their intended positions.
There are many examples of incompatible chemicals in dry reagent systems. For example, the base in white blood cell reagents causes premature hydrolysis of protease substrate. Iron in occult blood reagents causes premature oxidation of redox dye indicators to their colored form, which is also the result of the presence of iodate in glucose reagents. In the case of copper based tests for creatinine, the copper can oxidize redox indicators such as tetramethylbenzidine to their colored form in the absence of creatinine. Tests for occult blood in urine can be skewed by the presence of ascorbate in the urine test sample which acts as a reducing agent to cause false negative results and urine protein tests can be rendered inaccurate by the presence of buffers in the urine sample being tested. Dry assay devices for determining white blood cells in urine can be influenced by interference due to proteins in the urine sample and whole blood assays, such as blood glucose and blood CKMB, suffer from interference caused by red blood cells. In one embodiment, the present invention provides a means for alleviating these problems by separating two layers of a dry reagent device, at least one of which layers contains a reagent for detection of an analyte, with a test fluid permeable composition comprising a blend of an aqueous based polymer dispersion and a water soluble polymer, which blend has been cast and dried to form a layer having adhesive properties.
Previous methods for dealing with these problems have involved separating the reagents into discrete, stacked layers. There are, however, problems associated with the use of the discrete, stacked layer configuration. Thus, the top layer(s) must allow the test sample to pass to the lower layers while continuing to separate certain interfering chemicals and/or biochemicals. For example, metals such as copper or iron should be separated from redox indicators and bases from protease substrates. Oxidants such as iodate and reactants such as ascorbate need to be separated from redox indicators such as tetramethylbenzidine.
These problems are effectively dealt with by derivatizing the permeable composition of the present invention with elements which serve to remove interfering substances as they flow through the first layer of the device, through the permeable composition and into the device's second layer. This multi-layered format requires a permeable, adhesive material to hold the reagent layers together.
However, in the prior art, the contact between the layers was either insufficient to allow the reactants to pass from one layer to the adjacent layer when that was desired, or the reactants migrated from one layer to another when that was not desired.
There are various diffusable, adhesive compositions which can be used to secure two layers in integrated, multilayered reagent devices. Verbeck, in U.S. Pat. No. 3,993,451 uses adhesives to secure reagent containing particles to a substrate layer. The particles may be covered with a porous layer through which a component contained within a sample may pass to reach the reagent containing particles. In the device proposed by Verbeck, the adhesive is not used as a layer which separates reagent layers from detecting layers. Furthermore, the solid particles form separate detecting units which do not rely on movement of the reaction product with an analyte into an adjacent layer for detection.
Japanese Published Application 5-18959 A2 discloses the use of a hydrophobic polymer which does not swell in water as an adhesive to secure reagent layers and Japanese Published Application 5-26875 A2 discloses the use of a porous layer comprising a fluorine containing polymer as an adhesive to secure reagent layers. The polymers used in these Japanese systems are hydrophobic and consequently, they hinder rapid movement of sample fluids through the layers. For rapid testing, the sample fluid should pass through the layers of the device within less than one second. A water soluble adhesive would permit rapid movement of the sample fluid, but would cause the layers to separate as the adhesive begins to dissolve.
In EP 0 226 465 A2 a multilayer analytical device is described in which several porous sheets are bonded together with an adhesive placed so as to form openings through which liquids could pass. The adhesive itself was not capable of passing liquids so that openings were provided instead. The result being that not all of the available surface is useful and contact between the layers is not uniform.
The Greenquist '311 patent mentioned above also discloses a multilayer device for medical testing. Although the concept is valuable, in practice the multilayer device is not as satisfactory as would be desired. The layers must perform their intended function without interfering with the functioning of the adjacent layers. At the same time, the sample fluid must pass rapidly through the layers so that a result can be determined rapidly. Thus, the layers must act independently while not limiting the movement of the sample fluid. The present inventors have overcome these problems, as described below in a multilayer device and also in microfluidic devices.
In U.S. Pat. No. 4,824,640 a transparent layer is disclosed which is useful for containing analytical reagents which consists of a water soluble or water swellable component and an essentially insoluble film forming component. A similar layer is employed in U.S. Pat. No. 6,187,268 B1 as an overcoat over a dry reagent layer.
Dry reagent strips of the sort described above are not the only method of testing used near the patient. Microfluidic devices have been and are being developed which have advantages over multi-layered dry reagent strips. The general principles of certain microfluidic devices of interest to the present inventors is found in U.S. patent application Ser. No. 10/082,415. Microfluidic devices are designed to receive small liquid samples, e.g., blood and urine, and then process the samples through chambers interconnected by capillary passageways. The chambers may contain reagents which react with components in the sample as required for the intended analyses. The difficulties inherent in multi-layered test strips can be avoided. The needed reactions can occur sequentially, as the sample or portions of the sample are moved from one chamber to another, typically by capillary or centrifugal forces. Thus, as will be described in more detail below, the present invention may be applied in microfluidic devices in addition to multi-layered dry test strips.